Volume 5, Issue 1, January – 2020
ISSN No:-2456-2165
Determination of Mycobacterium Tuberculosis among
Sudanese Diabetic Patients by Cytokines and Compare
by Molecular Techniques in Khartoum State- Sudan
Dr. Wail Mohamed Osman Mohamed Ali Alkhraj1
Ahmed Kamal Bolad2
Atif Elagib3
Abstract:-
 Background
Pulmonary tuberculosis is the one of the main health
problem among Sudanese diabetic patients who are
considered as immune-compromised patients.
 Objectives of this study
To evaluate the different cytokines among study
groups ( diabetic and tuberculosis patients, non diabetic
tuberculosis patients, diabetic and cardio vascular
disease patients and healthy individuals). And detection
of Mycobacterium tuberculosis using Real Time PCR.
 Methodology
This study was designed as descriptive a
prospective, analytical case-control study. This study was
enrolled 402 individuals’ 40 were diabetic and
tuberculosis patients, 41 non diabetic tuberculosis ,
diabetic CVD were 160 and 161 healthy control. Five ml
of blood withdraw in plain vaccutainor after informed
consent from individuals whom participate in this study .
We measured the cytokines,(IL6 ,IL10 and TNFα) levels
from all participant using direct ELISA technique . Then
the Real time PCR was done for all participants.
 Results
The mean levels of IL6 was recorded in 161 healthy
persons 94.80. while recorded 196.27 in 41 tubercles non
diabetic patients 210.27. in 40 tuberculous diabetic
patients and 297.42 . in 160 patients with CVD, TB and
diabetic199.33. The mean levels of IL10 was recorded in
161 healthy persons mean 4.21. while recorded mean 8.84
in 41 tubercles non diabetic patients , mean 8.61 in 40
tuberculous diabetic patients and mean 7.90 in 160
patients with CVD, TB and diabetic.
The mean levels of TNFα was 81.59 pg/ml in 161
healthy persons. while it was 232.29 pg/ml in 40 tubercles
non diabetic patients , 261,78 in 40 tuberculosis diabetic
patients and 297.42 in 160 patients with CVD, TB and
diabetic. Real time PCR was positive among T.B patients
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International Journal of Innovative Science and Research Technology
and it was strong in 192 patients group ,while it was
negative It was a weak positive in 49 patients and
negative in 10 cardio vasulour disease. It was negative in
all healthy control 161 individules.
 Conclusion
TNF was elevated in all groups of tuberculosis
patients regardless they were diabetic or not. All T.B
patients showed positive result by Real time PCR .63 %
from CVD with T.B and DM showed positive result. The
estimation of cytokines with combination with real time
PCR showed strong correlation.
I. INTRODUCTION
A. Diabetes Mellitus
 Definition
Diabetes mellitus is not a single disease entity, but
rather a group of metabolic disorders sharing the common
underlying feature of hyperglycaemia (Mitchell et al., 2006).
 Incidence
Worldwide, over 140 million people suffer from
diabetes making this one of the most common diseases. In the
Western population the prevalence of DM has been estimated
to be 3-5% and the incidence is rapidly growing up and will
be more than doubled within 15 years. Type II DM accounts
for more than 80 % cases of DM and is slow-onset,
heterogeneous disorder, resulting from interactions between
environmental factors and polygenetic inheritance (Ostenson,
2001).
 TB and Diabetes
Diabetes is a chronic (long-lasting) disease that affects
how the body turns food into energy.Tuberculosis (TB) is a
serious health threat, especially for people living with
diabetes. Two TB-related conditions exist: latent TB
infection and TB disease. People with latent TB infection are
not sick because the body is able to fight the bacteria to stop
them from growing. People with TB disease are sick and
have active TB because the body cannot stop the bacteria
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from growing. People living with diabetes who are also
infected with TB are more likely to develop TB disease and
become sick with TB (CDC, 2019).
Someone with untreated latent TB infection and
diabetes is more likely to develop TB disease than someone
without diabetes. Without proper treatment, diabetes and TB
can increase health complications.
 In 2018, 9,029 new TB cases were reported in the United
States.
 In 2017, 20% of persons with TB in the United States also
had diabetes, as reported to the National TB Surveillance
System.
 30.3 million U.S. adults have diabetes.
 In the last 20 years, the number of adults diagnosed with
diabetes has more than tripled.
Untreated latent TB infection can progress to TB
disease. TB disease, without treatment, can progress from
sickness to death (CDC, 2019).
Fortunately, treatment options are available for people
with diabetes who also have either latent TB infection or TB
disease. If a person is diagnosed with TB infection, further
testing is required to rule out TB disease. People with either
latent TB infection or TB disease can be effectively treated.
Before beginning treatment for TB disease or for latent
TB infection, TB patients should talk to their doctor about
any other medication they are taking, including medicine for
diabetes. Some medications used to treat TB might interact
with medicine used treat diabetes (CDC, 2019).
 Impact of diabetes mellitus and tuberculosis on each
other:
There is strong evidence that DM increases the risk of
TB disease two- to three- fold. This association may be even
stronger in the presence of other risk factors. The increased
risk occurs in both type 1 DM and type 2 DM. However, type
2 DM accounts for over 95% of patients with DM worldwide
and therefore the public health burden of comorbid disease
from type 2 DM is much greater (WHO, 2006).
The increase risk of TB has mainly been described for
patients with smear-positive and culture-confirmed
pulmonary disease, with little published evidence so far
associating risk with extra-pulmonary TB (EPTB). There is
recent evidence to show that DM is an important risk factor
for MDR-TB (WHO, 2006).
 The effects of DM on response to TB treatment?
DM has several adverse effects on TB treatment.
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 Sputum bacteriological conversion:
There is some evidence that DM prolongs smear and
culture positivity at 2-3 months of treatment. Poor glycemic
control may be an important factor in this delay.
 Adverse drug reactions:
DM is probably associated with a higher risk of
hepatitis and renal drug toxicity. It is also associated with
gastrointestinal and other side effects that may overlap
between TB drugs and glucose lowering drugs used by
persons with DM.
 TB treatment outcomes:
DM adversely affects TB treatment outcomes. The
reasons are not completely understood but include the
immunosuppressive effects of DM itself, drug-drug
interactions, adverse effects from medications, suboptimal
adherence to medication, reduced bio-availability of the
drugs and other unlisted factors. The evidence points to an
almost doubling of the risk of death during TB treatment
among those with DM with the risk increasing to about five
times when adjustments are made for age and other potential
confounders.
Cardiovascular deaths could explain an increased rate
of deaths within months after starting TB treatment and the
much higher death rates among DM patients who smoke.
DM also increases the risk of TB treatment failure and
losses to follow-up. It is not clear whether the poorer TB
treatment outcomes described among those with worse
glycemic control are due to existing DM-related
complications or the hyperglycaemia itself. The risks of
relapse and recurrent TB in those who have completed anti-
TB treatment are also higher among those with DM
compared with those without: whether this is due to
reactivation of disease from the original Mycobacterium
tuberculosis or reinfection from another strain of
Mycobacterium tuberculosis is not known. Some preliminary
evidence suggests that improving glycemic control can lead
to better TB treatment outcomes and reduced risk of relapse
and recurrence ( WHO, 2011).
Tuberculosis is a widespread, and in many cases
fatal, infectious disease caused by various strains
of mycobacteria, usually Mycobacterium tuberculosis.
Tuberculosis typically attacks the lungs, but can also affect
other parts of the body. It is spread through the air when
people who have an active TB infection cough, sneeze, or
otherwise transmit respiratory fluids through the air. Most
infections do not have symptoms, known as latent
tuberculosis. About one in ten latent infections eventually
progresses to active disease which, if left untreated, kills
more than 50% of those so infected (Konstantinos, 2010).
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Tuberculosis (TB) has been still a major public health
problem in most developing countries and its incidence is
rising in many developed countries. TB is a re-emerging
infectious disease caused by a bacterium called
Mycobacterium tuberculosis (Konstantinos, 2010).
Cytokines are soluble, small proteins that are produced
by cells and act in a largely paracrine manner to influence the
activity of other cells. Currently, the term “cytokine”
describes proteins such as the tumour necrosis factor family,
the interleukins, and the chemokines. Virtually every
nucleated cell can produce and respond to cytokines placing
these molecules at the centre of most of the body’s
homeostatic mechanisms . Much of our knowledge of the
function of cytokines has been derived from studies wherein
homeostasis has been disrupted by infection and the absence
of specific cytokines results in a failure to control the disease
process. In this context, infection with Mycobacterium
tuberculosis (Mtb) has proven to be very informative and has
highlighted the role of cytokines in controlling infection
without promoting uncontrolled and damaging inflammatory
responses . Herein we focus on the key cytokine and
chemokines that have been studied in the context of human
TB using experimental medicine as well as Mtb infection of
various animal models, including non-human primates, mice
and rabbits. Perhaps the most important message of this
chapter is that in a complex disease such as tuberculosis (TB)
the role of any one cytokine cannot be designated either
‘good’ or ‘bad’ but rather that cytokines can elicit both
protective and pathologic consequences depending upon
context.
Why is TB such an informative probe allowing for
detailed investigation of the function of cytokines and
chemokines in immunity? One recent development in our
understanding of TB stems from theories of co-evolution
between modern humans and Mtb (Racquel et al., 2016).
B. Cytokines:-
 Tumor Necrosis Factor alpha (TNFα)
TNFα is a cytokine that is released following activation
of the immune system. Although it is primarily produced by
macrophages, TNFα can also be secreted by lymphocytes,
mast cells, endothelial cells, and fibroblasts. Because most
cells exhibit responsiveness to TNFα, it is considered a major
proinflammatory mediator (Racquel et al., 2016).
 The Interferons:-
The interferon family demonstrates the potential for
similar cytokines to play protective and pathological roles in
TB disease. Based on receptor specificity and sequence
homology, the interferons (IFNs) are classified into two
types. IFNγ is the only type II interferon and while
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International Journal of Innovative Science and Research Technology
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structurally related to the type I interferons IFNα and IFNβ,
these cytokine use different receptors and have distinct
chromosomal locations. Unlike type I IFNs that bind to a
common heterodimeric receptor comprised of IFNAR1 and
IFNAR2 chains, IFNγ binds to the IFNγ receptor (IFNGR)
which is comprised of two ligand binding IFNGR1 chains
that associate with two signal transducing IFNGR2 chains. In
addition while IFNγ is essential for survival following Mtb
infection the type I IFNs appear to be largely detrimental to
the host during TB and may be co-opted by the bacterium for
its own ends (Racquel et al., 2016).
 IL-12 Cytokine Family
The IL-12 family of cytokines belongs to the IL-6 super
family and is the only family composed of heterodimeric
cytokines and this unique feature bestows diverse and
pleiotropic functions due to promiscuous chain pairing . The
alpha chains of the IL-12 family (p19, p28 and p35) contain
four-helix bundle structures and pair with one of two beta
chains (either p40 or Epstein-Barr virus induced gene 3
(Ebi3)) (164–166). IL-12 is composed of the subunits
p35/p40, IL-23 of p19/p40, IL-27 of p28/Ebi3, and IL-35 of
p35/Ebi3 with expression of the distinct subunits being
regulated independently (166). In addition, IL-12p40 can also
be secreted both as a homodimer (IL-12p80 or IL-12p(40)2)
and as a monomer (IL-12p40) . Both macrophages and
dendritic cells are major producers of IL-12p40, IL-12, IL-23
and IL-27 . These cytokines are largely associated with the
induction and regulation of cytokine expression within
antigen-stimulated T cell populations (Racquel et al.,2016).
 Chemokines
Chemokines and cytokines are critical for initiating and
co-ordinating the organized and sequential recruitment and
activation of cells into Mtb-infected lungs. Correct
mononuclear cellular recruitment and localization are
essential to ensure control of bacterial growth without the
development of diffuse and damaging granulocytic
inflammation. An important block to our understanding of
TB pathogenesis lies in dissecting the critical aspects of the
cytokine/chemokine interplay in light of the conditional role
these molecules play throughout infection and disease
development (Tables I and II). Much of the data highlighted
in this chapter appears at first glance to be contradictory but
it is the balance between the cytokines and chemokines
which is critical and the ‘goldilocks’ (not too much and not
too little) phenomenon is paramount in any discussion of the
role of these molecules in TB. Determination of how the key
chemokines/cytokines and their receptors are balanced and
how the loss of that balance can promote disease is vital to
understanding TB pathogenesis and to identifying novel
therapies for effective eradication of this disease (Racquel et
al., 2016).
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Volume 5, Issue 1, January – 2020
 Detection and Quantification of Cytokines and Other
Biomarkers
Accurate measurement of cytokine concentrations is a
powerful and essential approach to the study of
inflammation. The enzyme-linked immunosorbent assay
(ELISA) is a simple, low-cost analytical tool that provides
both the specificity and sensitivity required for the study of
cytokines in vitro or in vivo. This communication describes a
systematic approach to develop an indirect sandwich ELISA
to detect and quantify cytokines, or other biomarkers, with
accuracy and precision. Also detailed is the use of sequential
ELISA assays to analyze multiple cytokines from samples
with limited volumes. Finally, the concept of a multiplex
ELISA is discussed with considerations given to cost and
additional time required for development (Evan et al., 2012).
Cytokines are a cornerstone of any study that deals with
inflammation, whether it is an in vitro cell culture system or
an in vivo animal model . The cytokine profile as a whole
and the relative abundance of one cytokine, and the
endogenous inhibitors, define an inflammatory process that is
in motion . Cytokines may be used to describe the nature of
the insult, infection, or injury , and may even be used to stage
the disease process (4). These studies revolve around the
ability to detect, quantify, and discriminate a single cytokine
from a multitude of biomolecules present in any given
sample. One such method that is routinely used is the indirect
sandwich enzyme-linked immunosorbent assay (ELISA)
(Evan et al., 2012).
The ELISA exploits the specificity of antibodies (Abs)
and uses them to capture and quantify an analyte of interest
from a given volume of sample, and it does this with
remarkable sensitivity (pg/mL or ~0.5 pM for a 15 kDa
protein) (Evan et al., 2012).
C. Polymerase chain reaction (PCR)
 Introduction
The polymerase chain reaction (PCR) is a technique
widely used in molecular biology. It drives its name from one
of its key components, a DNA polymerase used to amplify a
piece of DNA by in vitro enzymatic replication. As PCR
progresses, the DNA thus generated is itself used as a
template for replication. This sets in motion a chain reaction
in which the DNA template is exponentially amplified. With
PCR it is possible to amplify a single or few copies of a piece
of DNA across several orders of magnitude, generating
millions or more copies of the DNA piece. PCR can be
extensively modified to perform a wide array of genetic
manipulations. Almost all PCR applications employ a heat-
stable DNA polymerase, such as Taq polymerase, an enzyme
originally isolated from the bacterium Thermus aquaticus.
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ISSN No:-2456-2165
This DNA polymerase enzymatically assembles a new DNA
strand from DNA building blocks, the nucleotides, by using
single-stranded DNA as a template and DNA
oligonucleotides (also called DNA primers), which are
required for initiation of DNA synthesis. The vast majority of
PCR methods use thermal cycling, i.e., alternately heating
and cooling the PCR sample to a defined series of
temperature steps. These thermal cycling steps are necessary
to physically separate the strands (at high temperatures) in a
DNA double helix (DNA melting) used as template during
DNA synthesis (at lower temperatures) by the DNA
polymerase to selectively amplify the target DNA. The
selectivity of PCR results from the use of primers that are
complementary to the DNA region targeted for amplification
under specific thermal cycling conditions (Garibyan, 2013).
 Introduction to Real Time PCR
As the name suggests, real time PCR is a technique
used to monitor the progress of a PCR reaction in real time.
At the same time, a relatively small amount of PCR product
(DNA, cDNA or RNA) can be quantified. Real Time PCR is
based on the detection of the fluorescence produced by a
reporter molecule which increases, as the reaction proceeds.
This occurs due to the accumulation of the PCR product with
each cycle of amplification. These fluorescent reporter
molecules include dyes that bind to the double-stranded DNA
(i.e. SYBR® Green ) or sequence specific probes (i.e.
Molecular Beacons or TaqMan Probes). Real time PCR
facilitates the monitoring of the reaction as it progresses. One
can start with minimal amounts of nucleic acid and quantify
the end product accurately. Moreover, there is no need for the
post PCR processing which saves the resources and the time.
These advantages of the fluorescence based real time PCR
technique have completely revolutionized the approach to
PCR-based quantification of DNA and RNA. Real time PCR
assays are now easy to perform, have high sensitivity, more
specificity, and provide scope for automation. Real time PCR
is also referred to as real time RT PCR which has the
additional cycle of reverse transcription that leads to
formation of a DNA molecule from a RNA molecule. This is
done because RNA is less stable as compared to DNA(El-
Dawi et al., 2004)
 Rationale
Pulmonary tuberculosis is the one of the main health
problem among Sudanese diabetic patients who are
considered as immune-compromised patients.
This research used to facilitate the early detection of the
disease in order to prevent further serious complications of
tuberculosis. By the techniques of real time PCR in
combination with cytokines can aid to diagnose of pulmonary
tuberculosis which may play an important role in treatment.
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II. OBJECTIVES
A. General objective:-
 To evaluate the different cytokines among study groups
(diabetic tuberculosis patients, non-diabetic tuberculosis
patients, diabetic, and healthy individuals).
 To compare between different cytokines and Real Time
PCR in detection of Mycobacterium tuberculosis.
B. Specific objectives:-
 To compare IL-6, IL10 and TNFα between the following
groups:
 diabetic and healthy controls.
 diabetic with and without tuberculosis.
 patients with pulmonary tuberculosis and healthy
controls.
 diabetic patients with pulmonary tuberculosis and
diabetics without.
 diabetic with CVD and those without.
 poorly controlled and good controlled diabetics.
All compaition were done by using ELISA assay.
 To correlate the results of IL-6,IL10 and TNF with
duration of T2DB, by using SSPS, ONE way and Two
way ANOVA.
 To study the association of IL-6,IL10 and TNF with age
and gender.
 To confirms diagnostic tuberculosis by Real Time PCR.
 To compare between different cytokines and Real Time
PCR.
III. MATERIALS AND METHODS
A. Study design
This study was designed as a descriptive prospective,
analytical case-control study. Cross sectional hospital based
study. Which concerned to detect the cytokines (IL-6, IL10
and TNFα) by ELISA and detection of Pulmonary
Tuberculosis ( AAFB mycobacterium tuberculosis) by Real
time PCR. The tests of ELISA and PCR were performed for
all study groups.
B. Study area
This study was conducted in different hospitals (Alribat
Teaching hospital where located in the centre of Khartoum
and served the governmental persons and their relatives. Also
some patients were enrolled in the study from Jaber Abu
Elizz center for diabetic patients. Lastly Fadial private
hospital was involved. During the period from May 2015 to
Nov 2015.
C. Study duration
This study was started in September 2014 and ended in
2020
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D. Study population:-
 Sample size calculation
This study was enrolled 402 individuals’ 40 were
diabetic and tuberculosis patients, 41 tuberculosis and non-
diabetic ,tuberculosis diabetic and CVD were 160 and 161
healthy control. This number of patients was calculated
according to the equation of sample size determination which
expresses as follows.
 Sampling
Five ml of blood withdraw in plain vacationer after
informed consent from individuals to participate in this study.
The selected diabetic and non-diabetic subjects were aged
between 35-70 years.
E. Data collection
A coded enrollment number was given for each
enrolled patient. The data were collected by using a direct
interviewing questionnaire. Medical information was
collected from the patients with help of the physician. The
questionnaire was used to collect data regarding name, age,
gender, residence, duration of diabetes, presence of CVD
complications, history of systemic diseases and medication.
IV. RESULTS
 IL6 , IL10 and TNFα distribution of study groups:-
In the group statistics regarding both gender male and
female crossing the different agents
 TNF in female number 191 the mean = 199.91, std
deviation =180.716, std error = 13.076, in male number
209 the mean = 202.53, std deviation = 180.400, std error
= 12.479. the P. value in both genders was = 0.885.
 IL6 in female number 191 the mean = 165.81, std
deviation = 125.108, std error = 9.053, in male number
209 the mean = 151.99, std deviation = 116.699, std error
= 8.072. The P. value in both genders was = 0.254.
 IL10 in female number 191 the mean = 6.52, std deviation
= 4.684, std error = 0.339, in male number 209 the mean
= 6.64, std deviation = 6.321, std error = 0.437. The P.
value in both genders was = 0.833.
 HBA1C in female number 191 the mean = 7.25, std
deviation = 2.632, std error = 0.190, in male number 209
the mean = 7.19, std deviation = 2.582, std error = 0.179.
The P. value in both genders was = 0.833.
 This is one-way anova test: - P. value = 0.000 which is a
significant.
 Real time PCR result:-
It was positive among T.B strong 192 P.T ,while it was
negative in healthy control 161 patients ,weak positive 49
patients (figure -1 )
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TNF as dependent variable and PCR different results: -
 When PCR was positive in TB non-diabetic the mean =
389.87, std. deviation = 78.539, the number of
populations was 15.
 When PCR was positive in tuberculosis + diabetic the
mean = 386.71, std. deviation = 83.851, the number of
populations was 17.
 When PCR was positive in CVD + TB + Diabetic the
mean = 297.42, Std. Deviation = 198.423, the number of
populations was 160.
 Totally in TNF as dependent variable and PCR when
positive the mean = 312.55, Std. Deviation = 186.995, the
number of populations was 192.
 When PCR was negative in healthy the mean = 81.59,
Std. Deviation = 91.466, the number of populations was
161.
 Totally in TNF as dependent variable and PCR when
negative the mean = 81.59,
 Std. Deviation = 91.466, the number of populations was
161.
 When PCR was weak positive in TB non-Diabetic, the
mean = 141.38, Std. Deviation = 63.397, the number of
populations was 26.
 When PCR was weak positive in TB + Diabetic, the mean
=169.43, Std. Deviation = 59.888, the number of
populations was 23.
 Totally in TNF as dependent variable and PCR when
weak positive the mean =154.55, St. Deviation =62.747,
the number of populations was 49.
 Total results of TNF as dependent variable and PCR
different results in healthy the mean = 81.59, std deviation
= 91.466, the number of populations was 161.
 Total results of TNF as dependent variable and PCR
different results in TB non-Diabetic the mean = 232.29,
std deviation = 139.118, the number of populations was
41.
 Total results of TNF as dependent variable and PCR
different results in TB + Diabetic the mean = 261.78, std
deviation = 129.382, the number of populations was 40.
 Total results of TNF as dependent variable and PCR
different results in CVD +TB + Diabetic the mean
=297.42, std deviation =198.423, the number of
populations was160.
 Total results of TNF as dependent variable and PCR
different results the mean =200.79, std deviation =
180.029, the number of populations was 402.
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 IL6 as dependent variable and PCR different results: -
 When PCR was positive in TB non-diabetic the mean =
263.20, std.deviation = 91.793, the number of population
was 15.
 When PCR was positive in tuberculosis + diabetes the
mean = 266.00, std.deviation = 79.428, the number of
population was 17.
 When PCR was positive in CVD+TB+Diabetes the mean
= 199.33, std.deviation = 133.084, the number of
population was 160.
 Totally in IL6 as dependent variable and PCR when
positive the mean = 210.22, std.deviation = 128.401, the
number of population was 192.
 When PCR was negative in healthy the mean = 94.80,
std.deviation = 84.786, the number of population was 161.
 Totally in IL6 as dependent variable and PCR when
negative the mean = 94.80, std.deviation = 84.786, the
number of population was 161.
 When PCR was weak positive in TB non-diabetic the
mean = 157.65, std.deviation = 101.232, the number of
population was 26.
 When PCR was weak positive in tuberculosis + diabetes
the mean = 168.83, std.deviation = 73.552, the number of
population was 23.
 Totally in IL6 as dependent variable and PCR when weak
positive the mean = 162.90, std.deviation = 88.593, the
number of population was 49.
 Total results of IL6 as dependent variable and PCR
different results in healthy the mean = 94.80, std.deviation
= 84.786, the number of population was 161.
 Total results of IL6 as dependent variable and PCR
different results in TB non-diabetic the mean = 196.27,
std.deviation = 109.559, the number of population was 41.
 Total results of IL6 as dependent variable and PCR
different results in tuberculosis + diabetes the mean =
210.13, std.deviation = 89.481, the number of population
was 40.
 Total results of IL6 as dependent variable and PCR
different results in CVD+TB+Diabetes the mean =
199.33, std.deviation = 133.084, the number of population
was 160.
 Total results of IL6 as dependent variable and PCR
different results the mean = 158.23, St. Deviation =
120.719, the number of population was 402. Table (4.8.2)
 When PCR was positive in TB non-diabetic the mean =
263.20, std.deviation = 91.793, the number of population
was 15.
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Descriptive Statistics
Dependent Variable: IL6
PCR study population Mean Std. Deviation N
Positive TB non diabetic 263.20 91.793 15
tuberculosis + diabetes 266.00 79.428 17
CVD+ TB+ Diabstus 199.33 133.084 160
Total 210.22 128.401 192
Negative Healthy 94.80 84.786 161
Total 94.80 84.786 161
weak positive TB non diabetic 157.65 101.232 26
tuberculosis + diabetes 168.83 73.552 23
Total 162.90 88.593 49
Total Healthy 94.80 84.786 161
TB non diabetic 196.27 109.559 41
tuberculosis + diabetes 210.13 89.481 40
CVD+ TB+ Diabstus 199.33 133.084 160
Total 158.23 120.719 402
Table 1:-IL6 as dependent variable and PCR different results

 IL10 as dependent variable and PCR different results: -
 When PCR was positive in TB non-Diabetic the mean =
5.85, std.deviation = 2.289, the number of population was
15.
 When PCR was positive in tuberculosis + Diabetes the
mean = 8.05, std.deviation = 4.794, the number of
population was 17.
 When PCR was positive in CVD+TB+Diabetes the mean =
7.90, std.deviation = 5.085, the number of population was
160.
 Totally in IL10 as dependent variable and PCR when
positive the mean = 7.75, std.deviation = 4.913, the number
of population was 192.
 When PCR was negative in healthy the mean = 4.21,
std.deviation = 5.713, the number of population was 161.
 Totally in IL10 as dependent variable and PCR when
positive the mean = 4.21, std.deviation = 5.713, the number
of population was 161.
 When PCR was weak positive in TB non-Diabetic, the
mean = 10.56, Std. Deviation = 4.902, the number of
populations was 26.
 When PCR was weak positive in TB + Diabetic, the mean
= 9.02, Std. Deviation =3.887, the number of populations
was 23.
 Totally in IL10 as dependent variable and PCR when weak
positive the mean =9.84, St. Deviation = 4.477, the number
of populations was 49.
 Total results of IL10 as dependent variable and PCR
different results in healthy the mean = 4.21, std deviation
=5.705, the number of populations was 161.
 Total results of IL10 as dependent variable and PCR
different results in TB non-Diabetic the mean = 8.84, std
deviation =4.705, the number of populations was 41.
 Total results of IL10 as dependent variable and PCR
different results in TB + Diabetic the mean =8.61, std
deviation =4.265, the number of populations was 40.
 Total results of IL10 as dependent variable and PCR
different results in CVD +TB + Diabetic the mean = 7.90,
std deviation =5.085, the number of populations was160.
 Total results of IL10 as dependent variable and PCR
different results the mean =6.59, std deviation =5.579, the
number of populations was 402.

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Volume 5, Issue 1, January – 2020 International Journal of Innovative Science and Research Technology
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This is two-way anova test result.

Descriptive Statistics
Dependent Variable: IL10
PCR study population Mean Std. Deviation N
Pg/ml
Positive TB non diabetic 5.85 2.289 15
tuberculosis + diabetes 8.05 4.794 17
CVD+ TB+ Diabstus 7.90 5.085 160
Total 7.75 4.913 192
Negative Healthy 4.21 5.713 161
Total 4.21 5.713 161
weak positive TB non diabetic 10.56 4.902 26
tuberculosis + diabetes 9.02 3.887 23
Total 9.84 4.477 49
Total Healthy 4.21 5.713 161
TB non diabetic 8.84 4.705 41
tuberculosis + diabetes 8.61 4.265 40
CVD+ TB+ Diabstus 7.90 5.085 160
Total 6.59 5.579 402
Table 2:- IL10 as dependent variable and PCR different results

 TNF as dependent variable and PCR different results: -

When PCR was positive in TB non-diabetic the mean = 389.87, std. deviation = 78.539, the number of populations was 15.

Descriptive Statistics
Dependent Variable: TNF
PCR study population Mean Std. Deviation N
Pg/ml
Positive TB non diabetic 389.87 78.539 15
tuberculosis + diabetes 386.71 83.851 17
CVD+ TB+ Diabstus 297.42 198.423 160
Total 312.55 186.995 192
Negative Healthy 81.59 91.466 161
Total 81.59 91.466 161
weak positive TB non diabetic 141.38 63.397 26
tuberculosis + diabetes 169.43 59.888 23
Total 154.55 62.747 49
Total Healthy 81.59 91.466 161
TB non diabetic 232.29 139.118 41
tuberculosis + diabetes 261.78 129.382 40
CVD+ TB+ Diabstus 297.42 198.423 160
Total 200.79 180.029 402
Table 3:- TNF as dependent variable and PCR different results

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Volume 5, Issue 1, January – 2020 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
N Mean Std. Std. 95% Confidence Interval for Minimum Maximum
Deviation Error Mean

Lower Bound Upper Bound

Healthy 161 94.80 84.786 6.682 81.60 107.99 5 457

TB non diabetic 41 196.27 109.559 17.110 161.69 230.85 24 378

IL6 tuberculosis + diabetes 40 210.13 89.481 14.148 181.51 238.74 39 388

CVD+ TB+ Diabstus 160 199.33 133.084 10.521 178.55 220.11 9 476

Total 402 158.23 120.719 6.021 146.39 170.06 5 476

Healthy 161 4.21 5.713 .450 3.32 5.10 1 63

TB non diabetic 41 8.84 4.705 .735 7.35 10.32 3 19

IL10 tuberculosis + diabetes 40 8.61 4.265 .674 7.24 9.97 3 20

CVD+ TB+ Diabstus 160 7.90 5.085 .402 7.10 8.69 1 25

Total 402 6.59 5.579 .278 6.04 7.13 1 63

Healthy 161 5.00 .000 .000 5.00 5.00 5 5

TB non diabetic 41 5.00 .000 .000 5.00 5.00 5 5

tuberculosis + diabetes 40 9.80 1.896 .300 9.20 10.41 7 15

CVD+ TB+ Diabstus 160 9.34 1.928 .152 9.04 9.64 6 15

Total 402 7.21 2.602 .130 6.96 7.47 5 15

Table 4:- Descriptives all groups

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Volume 5, Issue 1, January – 2020
Fig 1:- Real time – PCR
This image showed the positive results above the blue
line. The lines in green that rose to the top are strong
positive.
V. DISCUSSION
The current study confirms that high levels of some
cytokines namely IL6, IL10 and TNFα constitute a diagnostic
biomarker for the latent tuberculosis in poorly controlled
diabetic. On the other hands, it confirms and extends the
findings of previous studies conducted outside Sudan
(Pakistan, India, United State, Iran and Sweden ).
 IL-6:-
The finding of this study that increased levels of IL6 in
poorly controlled diabetics is associated with positive
tuberculosis in these patients which is in agreement with the
finding of (Kiran et al,. 2016 ; Lavanya et al,.2015 ; Blanca
et al ,. 2008 ; Prati and Amit Goyal ,2013 ; Ponnana et al .,
2017 ; Fakhri et al., 2019 ; Emilie et al ,.2019 ; ). in the
studies conducted in Pakistan, India, United state ,Iran
Sweden respectively.
Kiran‫׳‬s and other (2016) study, found that the majority
of the study population who had diabetic and tuberculosis
have had high levels of IL-6, (study tested of 10 patients with
diabetes and 11 healthy endemic controls both with and
without MTB infection .The majority of patients tested
showed high levels of IL6. Denoting that they are infected of
mycobacteria tuberculosis .And this agreement with our
study.
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The India (Lavanya et al,.2015) study tested 150 active
pulmonary tuberculosis patients 190 household contacts ,
and 150 healthy controls. The majority of patients tested
showed high levels of IL6. Denoting that they are infected
of mycobacteria tuberculosis.
Another study in southern Texas (Blanca et al ,. 2008)
testet sixty-eight patients with tuberculosis . cytokine
responses were significantly higher in patients with
tuberculosis who had diabetes than in nondiabetic control
subjects. The effect was consistently and significantly more
marked in diabetic patients with chronic hyperglycemia
The majority of patients tested showed high levels of
IL6.
Another study in India newDelhi (Prati and Amit Goyal
,2013). The majority of patients tested showed high levels of
IL6.
A Similar study in Hyderabad in south India (Ponnana
et al ., 2017) stated cytokine genes associated with disease in
the household contact (HHCs) highlight their risk of
tendency towards the disease.
In a previous study in Iran (Fakhri et al., 2019) a total
of 105 smear-positive, including 78 newly diagnosed (ND)
and 27 under treatment (UT) patients with pulmonary TB and
111 age- and sex-matched healthy subjects were recruited.
ELISA cytokine assay was used to determine the plasma
levels of IL-6 plasma level was higher in the newly diagnosis
(ND) patients than healthy subjects and the UT patients.
1038
Volume 5, Issue 1, January – 2020
Another study in Stockholm Sweden (Emilie et
al,.2019) the study tested of active TB was detected in
54/161 (34%) of the study patients examined for suspected
TB. The patients were divided into groups according to
clinical and microbiological data; PTB 25, extra pulmonary
tuberculosis ( EPTB) 18, Clinical TB 11, Previous TB 22,
latent tuberculosis infection (LTBI) 62, TB negative 11 and
Other causes 12. The majority of patients tested showed high
levels of IL6.
In china (pane et al .,2019). A study included a total of
227 subjects consisting of active tuberculosis (ATB) patients,
latent tuberculosis infection (LTBI) individuals, and healthy
controls (HC). The majority of patients tested showed low
levels of IL6.This study is not in agreement.
 IL-10:-
Levels of IL-10 in the current study indicate IL10 is
possible diagnostic biomarker for the latent tuberculosis in
poorly controlled diabetic .On the other hands it confirms the
finding of previous studies conducted outside Sudan ( India,
United states, Sweden China , and Ghana ).
The finding in that increased levels of IL-10 in poorly
controlled diabetics is associate with positive tuberculosis in
these patients. This results were in agreement with the
finding of (Lavanya et al., 2015). Which was conducted in
India.
Another study in southern Texas (Blanca et al., 2008)
tested sixty-eight patients with tuberculosis. cytokine
responses were significantly higher in patients with
tuberculosis who had diabetes than in nondiabetic control
subjects. The effect was consistently and significantly more
marked in diabetic patients with chronic hyperglycemia.The
majority of patients tested showed high levels of IL10.
Another study in India newDelhi (Prati and Amit,
2013).Stated the majority of patients tested showed high
levels of IL10.
Another study in Stockholm Sweden (Emilie et al.,
2019).noted that active TB was detected in 54/161 (34%) of
the study patients examined for suspected TB. The patients
were divided into groups according to clinical and
microbiological data; PTB 25, EPTB 18, Clinical TB 11,
Previous TB 22, LTBI 62, TB negative 11 and Other causes
12. The majority of patients tested showed high levels of
IL10.
Another study in china (Bai et al., 2014) recruited 364
patients with type 2 diabetes mellitus and 677 healthy
controls. Patients carrying the -1082 GG genotype had a
significantly increased risk of type 2 diabetes mellitus. The
majority of patients tested showed high levels of IL10.
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In India (Nathella, 2015) a study tested a group of 88
individuals with PTB, 44 of whom had DM (PTB-DM) and
44 of whom had no diabetes (PTB). They also studied
another 88 individuals with LTB, 44 of whom had DM
(LTB-DM) and 44 of whom had no diabetes (LTB).plasma
Levels of IL-10 were significantly higher in PTB-DM
compared to PTB individuals.
Another study in India (Ramesh, 2015) tested 150 cases
presenting with diabetic neuropathy and 160 cases of age and
sex matched healthy controls were included in the study. The
results revealed that the chi- square test for heterogeneity for
IL-10 system was found to be significant.
In Ghana (Anthony ,2018) study tasted of eighty-three
pulmonary TB cases were used in the study. These included
49 MDR-TB and 34 DS-TB patients. This plasma level of
IL-10 was relatively higher than that of the pro-inflammatory
cytokines. The India (Lavanya et al,.2015) study tested 150
active pulmonary tuberculosis patients, 190 household
contacts and 150 healthy controls. The IL-10 levels were low
in APTB compared to HHC and HCs and no significant.
In china (pane et al., 2019) a study included a total of
227 subjects consisting of active tuberculosis (ATB) patients,
latent tuberculosis infection (LTBI) individuals and healthy
controls (HC). The majority of patients tested showed low
levels of IL6.this study no agreement.
 TNF alpha :-
Levels of TNFα in the current study indicates TNFα is a
possible diagnostic biomarker for the latent tuberculosis in
poorly controlled diabetic patients. On the other hands it
confirms the finding of previous studies conducted outside
Sudan ( Pakistan ,India, United states, Sweden, China , and
Ghana). The finding is that increased levels of TNFα in
poorly controlled diabetics is associated with positive
tuberculosis in these patients.
This results was in agreement with finding of similar
(Kiran et al., 2016 ;Lavanya et al,.2015). in that studies
conducted in Pakistan and India respectively. The Pakistan
(Kiran et al., 2016) study tested of 10 patients with diabetes
and 11 healthy endemic controls both with and without MTB
infection .The majority of patients tested showed high levels
of TNFα. Denoting That they are infected of mycobacteria
tuberculosis.
The India (Lavanya et al., 2015) study tested 150 active
pulmonary tuberculosis patients,190 household contacts and
150 healthy controls The median values of TNF-α cytokine
were significantly high among APTP. Another study in
southern Texas (Blanca et al., 2008) tested sixty-eight
patients with tuberculosis . cytokines responses were
significantly higher in patients with tuberculosis who had
diabetes than in nondiabetic control subjects. The effect was
1039
Volume 5, Issue 1, January – 2020
consistently and significantly more marked in diabetic
patients with chronic hyperglycemia. The majority of patients
tested showed high levels of TNFα this results proved our
study.
Another study in Stockholm Sweden (Emilie et al
,.2019) noted that active TB was detected in 54/161 (34%) of
the study patients examined for suspected TB. The patients
were divided into groups according to clinical and
microbiological data; PTB 25, EPTB 18, Clinical TB 11,
Previous TB 22, LTBI 62, TB negative 11 and Other causes
12). The majority of patients tested showed high levels of
TNFα. And this is another agreement with our study.
In china (pane et al., 2018) study included a total of 227
subjects consisting of active tuberculosis (ATB) patients,
latent tuberculosis infection (LTBI) individuals and healthy
controls (HC). The majority of patients tested showed high
levels of TNFα.this study was in agreement.
In Ghana (Anthony ,2018) eighty-three pulmonary TB
cases were used in the study. These included forty-nine
MDR-TB and thirty-four DS-TB patients. This plasma level
of TNFα was relatively higher than that of the pro-
inflammatory cytokines. In India (Ramesh ,2015) study
tested 150 cases presenting diabetic neuropathy and 160
cases of age and sex matched healthy controls were included
in the study. The results revealed that the chi- square test for
heterogeneity for TNFα not significantly associated with
development of Diabetic Neuropathy.
VI. CONCLUSION
 Serum levels of IL6 ,IL10 and TNFα increased in
patients with no signs and symptoms of tuberculosis
(latent phase ).
 The cut off values 68pg/ml ; 5.85pg/ml; 297.42pg/ml for
IL6; IL10 and TNFα respectively are diagnostic for latent
tuberculosis.
 There was no relationship between Gender and IL6, IL10
and TNFα levels.
RECOMMENDATION
 All patients with complicated diabetic should be test for
the latent tuberculosis in poorly controlled diabetic
mellitus type Ⅱ,
 Implementation of cytokines of IL6 , IL10 and TNFα as
screening and complementary test with real time PCR for
detection of latent tuberculosis in poorly controlled
diabetic .
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